Synthetic toxin structures have been designed in attempts to create new therapeutics that exhibit cell-targeted cytotoxicity after administration to a patient (see, Pastan I et al., Annu Rev Med 58: 221-37 (2007), for review). The aim of such molecular engineering is to design cytotoxic molecules comprising at least two functional domains: a cell-targeting region and a cytotoxic toxin region. A common type of such cytotoxic molecules is chimeric proteins comprising both a cell-targeting region and a toxin-derived cytotoxic region. One way to design cytotoxic chimeric proteins is to identify promising candidates or molecular frameworks by high-throughput screening of molecular libraries using technologies to select for desired characteristics, such as, e.g. high affinity target binding and/or cell internalization (see Poul M et al., J Mol Biol, 301: 1149-61 (2000); Levin A, Weiss G, Mol Biosyst 2: 49-57 (2006); Mazor Y et al., J Immunol Methods 321: 41-59 (2007); Bidlingmaier S et al., Cancer Res 69: 1570-7 (2009); Zou Y, Marks J, Methods Enzymol 502: 43-66 (2012)).
The purpose of molecular library screening is to search among a large diversity of molecules for those molecules with desired properties or functions. In particular, protein display technologies enable statistically powerful, high-throughput screening of large protein libraries in order to identify polypeptides which exhibit desired properties, such as specific molecular interactions (see, Glöckler J et al., Molecules 15: 2478-90 (2010), for review). For example, various protein display screening methods have been used to screen for protein-ligand interactions, such as bacteriophage display, bead-surface display, cell-surface display (prokaryotic or eukaryotic), RNA display, protein-DNA linkage display, ribosome display, and virus display. In particular, in vitro display technologies have become powerful tools for identifying immunoglobulin-type domains that bind human proteins for biomedical applications (Bradbury A et al., Nat Biotechnol 29: 245-54 (2011)).
In vitro display methods called bacteriophage display (phage display) became standard as early as 2005 (Hoogenboom H, Nat Biotechnol 23: 1105-16 (2005)). In particular, phage display screening of immunoglobulin domains was a major technological breakthrough (see McCafferty J et al., Nature 348: 552-4 (1990)). Phage display methods enabled the screening of relatively large polypeptide libraries (e.g. greater than 1×109 unique library members) and improved screening power. Later, the development of cell-free, in vitro protein display systems like RNA display, ribosome display, and protein-DNA linkage systems enabled widespread screening of even larger libraries (e.g. 1×1015 unique library members).
Screening molecular libraries by protein display involves the en masse expression and screening of members of a protein library while maintaining a physical connection between phenotype and genotype, i.e. the displayed protein is physically linked to the nucleic acid encoding that individual protein. For example, 1) in phage display—each library polypeptide is part of the outer capsid of a phage particle containing genetic material encoding that specific polypeptide, 2) in yeast display—each library polypeptide is designed to form part of the outer cell wall of a yeast cell containing genetic material encoding that specific library polypeptide, 3) in ribosome display—each library polypeptide remains tethered to a ribosome physically associated with an RNA encoding that specific polypeptide, and 4) in RNA display—each library polypeptide is covalently coupled to an RNA encoding that specific polypeptide (Valencia C et al., Methods 60: 55-69 (2013)). The physical linkage of genotypes and phenotypes enables the screening of specific molecular interactions en masse while maintaining a connection between each library member and its individual genotype for identification of the biological sequence(s) associated with the desired phenotype(s).
Protein display technologies such as phage display, bacterial display, yeast display, RNA display, and ribosome display have all been widely used to screen for desired molecular affinity interactions (Chen T, Keating A, Protein Sci 21: 949-63 (2012)). Protein display screening may also be used to identify polypeptides with other characteristics or functions, such as cell binding and cellular internalization (Poul M et al., J Mol Biol, 301: 1149-61 (2000); Nielsen U et al., Biochim Biophys Acta 1591: 109-18 (2002); Liu B et al., Cancer Res 64: 704-10 (2004); Levin A, Weiss G, Mol Biosyst 2: 49-57 (2006); Mazor Y et al., J Immunol Methods 321: 41-59 (2007); Bidlingmaier S et al., Cancer Res 69: 1570-7 (2009); Zhou Y et al., J Mol Biol 404: 88-99 (2010); Zhou Y et al., Arch Biochem Biophys 526: 107-13 (2012); Zhou Y, Marks J, Methods Enzymol 502: 43-66 (2012)).
Chimeric fusion proteins represent one class of molecule that can be discovered and/or improved using protein display screening. Cytotoxic fusion proteins derived from certain toxins depend in part on a potent ribotoxicity not present in most proteins. Commonly, cytotoxic fusion proteins are derived from naturally occurring protein toxins which comprise a ribotoxic enzymatic domain (Pastan I, et al., Annu Rev Med 58: 221-37 (2007); Potala S, et al., Drug Discov Today 13: 807-15 (2008); Kreitman R, Bio Drugs 23: 1-13 (2009)). For example, certain toxins catalytically inhibit ribosome function, such as, e.g., ribosome inactivating proteins (RIPs) with ribosome N-glycosidase activity, including ricins, sarcins, Shiga toxins, abrins, gelonins, and saporins, and, e.g., bacterial AB toxins with ADP-ribosylation activity, including diphtheria toxins and Pseudomonas exotoxins (Shapira A, Benhar I, Toxins 2: 2519-83 (2010)). The cytotoxic mechanisms of all these toxins are based on the ability to inhibit protein translational via damaging ribosome function, i.e. ribotoxicity.
It is desirable to use protein display screening to discover and develop cytotoxic chimeric proteins comprising toxin-derived ribotoxic regions, such as, e.g., immunotoxins, ligand-toxin fusions, and immuno-RNases. Protein display screening may be used to identify novel chimeric cytotoxic polypeptides and/or optimize any selectable characteristic of a cytotoxic polypeptide, such as, e.g., target molecule binding affinity, cell targeting, cell binding, cellular internalization, subcellular routing, enzymatic activity, and cytotoxicity. However, effective protein display screening of libraries comprising toxin-derived polypeptides can be disrupted by the ribotoxic effects of toxin-derived polypeptide regions.
Protein display screening can be hindered by ribotoxicities present in expression libraries comprising toxin-derived ribotoxic regions. To work around this problem, ribotoxic polypeptides have mostly been developed in a piecemeal fashion by screening cell-targeting domains in the absence of any toxin-derived domain and then linking the two domains together to form cytotoxic chimeric molecules. Alternatively, in the few rare instances where protein display screening of ribotoxic polypeptides has been successful, success was only possible with relatively small libraries (e.g. ˜1×104 and ˜4×105) capable of significantly less power than possible using routine screening methods available for polypeptide expression libraries lacking toxin-derived ribotoxic domains (Cheung M et al., Mol Cancer 9: 28 (2010); Cizeau et al., J Biomol Screen 16: 90-100 (2011)).
Because most chimeric fusion proteins comprising ribotoxic regions have been developed in a piecemeal manner, with the cell-targeting region isolated separately from the ribotoxic region, this has resulted in the need for additional molecular engineering steps to build the complete chimeric structure, which might then acquire different physical and functional attributes. Moreover, the extra step of completing the chimeric structure by adding the cytotoxic component represents an additional inefficiency in the development process. Furthermore, even if the chimeric structure retains the desired functional activities of its components, the production process for making the final, cytotoxic, chimeric structure may require additional optimization steps which were not apparent when producing the cell-targeting and ribotoxic domains independently. For all these reasons and perhaps others, the current approaches of designing and producing toxin-derived, ribotoxic fusion proteins has commonly led to the selection of molecules with less than ideal properties (Weldon J, Pastan I, FEBS J 278: 4683-700 (2011)).
There remains a need in the art for methods of display screening libraries comprising toxin-derived, ribotoxic polypeptides in protein display formats which are more effective, more efficient, more statistically powerful, and minimize unwanted selection biases in order to more efficiently identify and select for ribotoxic proteins and polypeptides with more desirable properties such as, e.g., high-affinity, target-cell binding, promotion of cellular internalization, and ease of production.